Metabolism of Chlorophyll in Higher Plants V. Chlorophyll Biosynthesis in Nicotiana Callus

Metabolism of Chlorophyll in Higher Plants v. Chlorophyll Biosynthesis in Nicotiana Callus

KEIKO KATO and SEKI SHIMIZU

Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Tokyo 112, Japan

Received July 29,1981 . Accepted August 31,1981

Summary

The callus of Nicotiana ingulba has the unusual ability to produce little or no chlorophyll on exposure to light. The activities of the enzymes involved in the early stage of ChI biosynthesis - ALA synthesizing activity, ALAD and PBGase - were studied. ALA synthesizing activity was detected in non-chlorophyllous as well as chlorophyllous callus. When HC labelled ALA was fed to the callus, 14C was incorporated into ChI in green callus and into CO2 in white callus. These results suggest that the ChI content of callus is regulated by the activities of ALAD and PBGase. The biosynthetic pathway of ALA is also discussed.

Key words: Nicotiana ingluba callus, chlorophylls, 8·aminolevulinic acid, 8·aminolevulinic acid dehydratase, porphobilinogenase.

Introduction

Although the totipotency of cultured plant cells has been confirmed by many workers, in many cases the plant cells synthesized little or no chlorophyll (Chi) and could not grow autotrophically under normal conditions. Much effort has been directed towards finding the correct conditions for culturing plant cells autotrophically and some success in culturing them under CO2-enriched and full light conditions has been reported (Berlyn et al., 1978; Sato et al., 1979). Little, however, is known about the process controlling Chi biosynthesis at low levels.

In the present study, we give attention to the much lower ChI content of callus cells than that of leaf cell. In particular, we studied Chi biosynthesis in Nicotiana plant callus, particularly the early stages of the biosynthetic pathway from cS-aminolevulinic acid (ALA) to Uroporphyrinogen (Urogen) III. ALA is the first specific precursor of tetrapyrrole compounds including Chl. In animal tissues and bacteria, ALA is produced via the «classical pathway» [glycine + succinyl-CoA ~ ALA, catalyzed by ALA synthetase (ALAS)]. Recent works have shown that this pathway also

Abbreviations: ALA, 8-aminolevulinic acid; ALAD, ALA dehydratase; ALAP, ALA pyrrole; ALAS, ALA synthetase; ChI, chlorophyll; DOVA, y-8-dioxovaleric acid; DTT, dithiothreitol; LA, levulinic acid; PBG, porphobilinogen; PBGase, porphobilinogenase; Urogen III, uroporphyrinogen III.

z. Pjlanzenphysiol. Bd. 104. S. 409-418. 1981.

410 KEIKO KA TO and SEKI SHIMIZU

operates in plants and green algae (Hampp et aI., 1975; Hampp and Wellburn, 1976; Hendry and Stobart, 1978; Klein and Senger, 1978 a, b). However they could not detect ALA synthetase activity. An important exception to this is the formation of ALA from glutamate or a-ketoglutarate by a series of reactions probably involving y-8-dioxovaleric acid (DOVA) as intermediate (Beale, 1978; Beale and Castelfranco, 1974; Castelfranco and Schwarcz, 1978; Gough and Kannangara, 1977; Kannangara and Gough, 1977; Lohr and Friedman, 1976) in green plants (the C-5 pathway).

ChI biosynthesis in green plants, chlorella and blue-green algae has been shown to be regulated at two points (Kasemir and Masoner, 1975); (i) the ALA forming system(s) and (ii) the ALA metabolizing enzymes. Concerning ChI biosynthetic activity of callus cells, Schneider (1973) reported that ALAD and PBGase activities of the green callus of N. tabacum increased in parallel with ChI content. In peanut suspension culture, ALA synthesizing activity regulates tetrapyrrolic compound synthesis (Van Huystee, 1977).

In the present work, we compare the ChI synthesizing activities of non-chlorophyllous and chlorophyllous callus. ChI synthesizing activity was determined at the following stages: (I) The overall activity of ChI synthesis. 14C-Iabelled ChI precursor compounds were given to callus cells and the radioactivity of the formed ChI was determined. (II) ALA synthesizing activity. In the presence of levulinic acid (LA), a competitive inhibitor of ALAD, callus cells accumulate ALA. It is considered that under the condition the amount of ALA suggests an ALA synthesizing activity (Beale, 1971). (III) Activity of ALAD -+ formation of porphobilinogen (PBG). The second intermediate in ChI biosynthesis, PBG is formed by the condensation of two molecules of ALA, catalyzed by ALAD. (IV) Activity of PBGase (the complex of uroporphyrinogen (Urogen) I synthetase and Urogen III cosynthetase) -+ formation of the first cyclic tetrapyrrole intermediate, Urogen III.

Materials and Methods

Plant materials and culture conditions

Two strains of callus derived from Nicotiana ingulba were used: Chlorophyllous callus (Gcallus), kindly supplied from the Institute of Biological Science, University of Tsukuba and non-chlorophyllous callus (W-callus), derived from seed in our laboratory. G-callus and Wcallus were isolated in 1970 and 1972 respectively and have been maintained on Linsmaier and Skoog medium (Linsmaier and Skoog, 1965) supplemented with 3 % sucrose, 0.5 ppm 2,4-D, 0.25 ppm kinetine and 1 % agar. The cultures were grown at 27 ± 1 °C under continuous fluorescence light (average intensity, 4,000 lux) or in the dark. Subcultures were routinely made every 28 days.

Biochemicals

[2-14C]glycine (specific activity, 196-207 . 107 Bq mmol- I), [4-!4C]ALA (specific activity, 218 . 107 Bq mmol- I

) were obtained from the Radiochemical Center, Amersham, England. [U-14C]a-ketoglutarate (specific activity, 1082 . 107 Bq mmol- I), [5-14C]ALA (specific activity, 49185' 107 Bq mmol- I

) were obtained from New England Nuclear, and

Z. Pjlanzenphysiol. Bd. 104. S. 409-418. 1981.

Chlorophyll biosynthesis in Nicotiana callus 411

[U-HC]glutamate (specific activity, 170-226 . 107 Bq mmol- 1) and [3,5-HC]glutamate (specific activity, 19 - 57 . 107 Bq mmol- 1) from Commissariat a L'Energie Atomique, Saclay, France.

ALA and PBG were obtained from Sigma Chemical Co., St. Louis.

Preparation 0/ crude enzyme extract

Crude enzyme was extracted from an acetone pulp of callus of N ingluba. The acetone pulp was prepared as described (Loomis, 1959) and proteins were extracted with 0.1 M Tris-HCI buffer (pH 8.2), containing 1 mM DTT (30 ml buffer per 1 g pulp). This extract was centrifuged at 11,000 xg for 20 min and the supernatant was concentrated by ultrafiltration with PM 10 membrane (Amicon, Co., Lexington, U.S.A.) for enzyme assay.

Assay 0/ enzymes

ALAD activity in the crude enzyme extract was assayed by the method of Mauzell and Granick (1956) and PBGase activity by the method of Schneider (1971).

Isolation and determination 0/ ALA

3 g of 10-day-old dark grown callus were suspended in 5 ml of 0.01 M Na-phosphate buffer (pH 6.0) containing 5 mM of LA under steril conditions. During incubation in the light at 29°C with moderate shaking, the cells were harvested at periodic intervals and ALA was extracted. The amount of ALA was determined by the metod of Beale and Castelfranco (1974).

Incorporation o/I'C-Iabelled compounds into Chi

3 g of 10-day-old cells were suspended in 5 ml of 0.Q1 M Na-phosphate buffer (pH 6.0) containing 1 mmole of 1'C-Iabelled compounds (s. a., 19 . 10' Bq mmol- 1) listed in Table 1. The cell suspension was incubated in a 30 ml incubation flask which was compartmented into the main compartment and the center well. The suspension was introduced into the main compartment and the center well contained a strip of filter paper which had been soaked with 0.05 ml of 20 % KOH. The incubation was conducted with shaking in the continuous light (4,000 lux) at 29°C.

Extraction and separation 0/ the HC-Iabelled pigments

After 6 h of incubation, the cells were harvested, washed with water and then extracted with 80 % acetone three times. The combined acetone extracts were mixed by shaking with an equal volume of ether in a separatory funnel. Fifteen % NaCI solution was added to form two layers. The ether phase was dehydrated with Na2S0. and brought to dryness in vacuo. The residue was dissolved in a small volume of ether and applied to a TLC plate (silic gel-H, Merck).

TLC of pigments was run in the dark with two solvent systems successively in the same direction; (1) petroleum ether (b.p. 50-70°) - isopropanol - H20 (100: 10: 0.25, v/v) and (2) hexane - acetone (60: 40, v/v). A good separation of ChIs and carotenoids was obtained on the chromatogram. After development, the red fluorescent bands corresponding to the pigments were viewed under UV light. The chromatograms were also scanned for radioactivity using a radiochromatograms scanner (Aloka, JTC-201, Japan). The pigments were scraped off and dissolved in 7 ml of scintillation cocktail (500 g toluene containing 0.23 g PPO) in a vial. Radioactivity was determined using a tri-carb liquid scintillation spectrometer (Packard, model 3255).


412 KEIKO KATO and SEKI SHIMIZU

Cell growth

The growth of callus was followed by the determination of fresh and/or dry weight of cells at appropriate intervals. The cells were collected by filtration through a layer of Miracloth (Calbiochem-Behring Corp.) on a funnel under reduced pressure and the fresh weight was determined. Dry weight was determined after drying the cells in an oven at 80°C for 24 hrs.

Determination 0/ ChI and protein contents

ChI content of a 80 % acetone extract of callus was determined according to the method of Arnon (1949). Protein was estimated by the method of Lowry et al. (1951) using serum albumin as a standard.

Results

Growth characteristics

Fig. 1 shows the growth curves of G- and W-callus grown under continuous light. The dry weight was determined every 5 days for a period of 40 days. W- and G-callus grew exponentially from the start of culture till around day 20. After that period, two levels of growth were distinguishable. The growth rate in the latter period was higher with W-callus. The G-callus grew slowly and reached a stationary phase at about day 25, but consistent growth was observed after 25 days with W-callus.

Chlorophyll content

Changes in ChI content for the two callus cultures are shown in Fig. 2. With illuminated G-caIlus, accumulation began very soon after a subculture was established.

30

.'

d I . .

Days of incubation

Fig. 1: Growth curves of N. ingluba callus. Changes in fresh weight per flask of (-e-) Gand of (-0-) W-calluses.



The pigment content reached a maximum value of 90-100 J.lg . g-l fr.w., by day 14. When the culture was extended beyond 2 weeks, the ChI content decreased sharply and the cells turned brown. Therefore 10 -14 days callus, which actively synthesized ChI, was used in the present study. W-callus produced scarcely appreciable amounts of ChI (below 5 J.lg . g-l fr.w.).

G

80 40

G G

60 30 G 15

G

G G

40 20 G 10

<:: G ~ 2 'c c: :J

G li 0 G v Cl c :c <t G G ~ ...J U «

G 20 10 G 5

G

0 0

Days of incubation

Fig. 2: Changes in the Chi content, and ALAD and PBGase activities during the growth of N. ingluba callus. Chi content: J.lg . g-l fresh weight (D), ALAD unit: nmol PBG . mg- 1

•

protein' h- 1 (c:J), PBGase unit: nmol pyrrole . mg- 1 protein' h- 1 (I11III).

Changes in Chl synthetic activity

ALAD and PBGase Changes in ALAD and PBGase activities in the two kinds of callus cultures are shown

in Fig. 2. With G-callus, ALAD and PBGase activities show similar curves; both activities increased at the beginning of subculture and lasted for 14 days, and thereafter the activity fell steadily with time. By contrast, there was little fluctuation in the activity of the two enzymes with W-callus.

The peaks of the enzyme activity with the illuminated G-callus coincided with the peak in the ChI content during the early S-phase of growth.

Incorporation o/14C-ALA into Chlorophyll

Incubation of 10-day-old callus cells with 14C-Iabelled precursor compounds led to 14C-Iabelled ChI formation. As shown in the scanning radio chromatogram of

Z. Pjlanzenphysiol. Bd. 104. S. 409-418.1981.


G. callus (Fig. 3, I), 14C-ALA was incorporated into pheophytins a and b, ChI a and b and chlorophyllide, but not into carotenoids. Phaeophytins and Chlorophyll ide might be formed from ChI a and b during the extracting and separating procedures, thus the values of 14C incorporation into ChI in Table 1 was the sum of the radioactivity of ChI a and b, pheophytins a and band chlorophyllide. In W-callus, however, there was no recognizable peak in the chromatogram. .

~t~. ~ 0 t ~400 G .., u g300

'8 a: 200

100

Frqnt

TJ" ~ i Ch ide

o 1111 ~ II

Chlb Chi. PhbPh. Carot.

Fig. 3: (I) Radiochromatogram,of the ether soluble pigments from the illuminated N ingluba callus incubated with HC-ALA. W: with W-callus, G: with G-callus. - (II) Thin-layer chromatogram of the same pigments. Green pigments @, yellow pigments O.

Table 1: Incorporation of 14C-labelled precursor into chlorophyll by the illuminated N. ingluba callus.

14C-Compounds Samples Total uptake, % 14C02, % of 14C-chlorophyll, of applied label total uptake % of total uptake

[2_14C] Gly G 14.10 3.87 1.00 W 34.80 3.16 0.421

[3,4 - 14C] Glu G 8.53 12.60 0.270 W 6.33 13.10 0.046

[U - 14C] u-KG G 25.80 82.10 0.105 W 59.90 46.90 0.074

[4_14C] ALA G 6.44 2.62 19.80 W 6.69 16.40 0.512

[5- 14C] ALA G 6.59 3.56 22.80 W 7.96 14.10 0.32

Results are given as the average of 3 experiments.



In Table 1, with G-callus about 20 % of uptaken 14C taken up from ALA was incorporated into ChI, indicating that the exogenous 14C-ALA was directly used for ChI synthesis. In contrast, W-callus poorly incorporated 14C-ALA into ChI, but evolved 14C02 to a remarkable degree. These results seemed to indicate that with W-callus exogenous ALA was preferentially metabolized by other metabolic pathway(s). [2-14C]glycine, [U-14C]a-ketoglutarate or [3,4- 14C]glutamate, which are thought to be the precursors of ALA (Beale and Castelfranco, 1974; Hampp et aI., 1975; Hampp and Wellburn, 1976; Hendry and Stobart, 1978; Porr and Grimme, 1974), were not incorporated into ChI by both callus with the exception of glycine.

ALA synthesizing activity

Because little ALA accumulates in normal green plant tissues, the indirect method of Beale and Castelfranco (1974) using LA as a competitive inhibitor of ALAD was employed for the determination of ALA synthesizing activity. After the examination of the effect of LA concentration on cell growth, ChI content and ALA accumulation, 5 mM was chosen as the standard concentration in subsequent experiments.

The rate of ALA production was much higher in W-callus than in G-callus by day 4 (Fig. 4). After that time the rate of ALA production in W-callus sharply decreased.

120

100 1, ~ 80 (5 E ". <{ 6 ....J <{

, , , :' " , , ,

---_ .. ----------' o 2 3 4 5 6 7 8 days

Days of incubation

Fig. 4: Change in ALA synthesizing activity during the growth of N. ingluba callus. The accu· mulation of ALA by the illuminated (-0-) G- and (-e-) W -calluses in the presence of LA. G-callus produced a small amount of Chi nevertheless in the presence of LA. Total ALA synthesizing activity of G-callus, ALA accumulated plus 8 times of Chi formed, was also shown ( - - - e ---).

The rate of ALA production in G-callus attained a steady level by day 4 and was followed by a marked decrease after the 6th day. During this period, the inhibition of ALAD by LA became insufficient and a small amount of ChI was recognized in the


416 KEIKO KA TO and SEKI SHIMIZU

LA-treated callus. Therefore the total ALA synthesizing activity (moles of ALA accumulated plus 8 times moles of Chi formed) was calculated.

Discussion

Under the present conditions, the greening of G-callus was completed by day 14 under continuous light (Fig. 2). Compared with the Chi content of field grown N ingluba leaves, 85.8 J..lg . g-l fresh weight, the Chi content of G-callus was 1/20 as much as leaf. The activity of the enzymes per mg of protein was 1/10-1/2 as much as leaf. If enzyme activity was expressed as moles of product per g of Chi, the values of G-callus were rather higher than those of green leaf.

After the 14th day, Chi content on a fresh callus basis began to decrease, and ALAD and PBGase activity of the callus also began to decrease. When the activity of both enzymes was plotted against Chi content, it remained almost unchanged during the course of growth. This indicates that there is a regulatory system for ALAD and PBGase activity which might participate in regulating Chi synthesis.

The accumulation of ALA in LA-treated non-chlorophyllous callus suggested that ALA was synthesized to the same extent as in chlorophyllous callus. W-callus cells, however, were unable to use the formed ALA for Chi synthesis efficiently, because activities of ALAD and PBGase were remarkably low in non-chlorophyllous cells. So the excessive ALA might be metabolized in a metabolic pathway(s) other than the Chi synthesis pathway. This speculation was supported by the 14C-ALA feeding experiment. 14C-ALA which was fed to G-callus cells was incorporated into Chi with high efficiency (about 20 %), but if fed to W-callus cells 14C was poorly incorporated into Chi and appeared as CO2 in a good yield (about 15 %) (Table 1). No noticeable changes in amino and organic acid composition of callus cells owing to ALA feeding were recognized (unpublished data). These results indicate that ALAD andlor PBGase activity controls Chi synthesis in Nicotiana callus and that the cause of the reduced Chi synthesis with W-callus was probably due to the low activity of its ALAD and PBGase activities.

Porra and Grimme (1974) reported that the Chi synthesis of Chlorella fusca is regulated at the step of ALA formation. Our studies, however, indicated that Chi synthesis in N ingluba callus was limited by the activity of ALAD andlor PBGase. This is consistent with those of N tabacum callus (Schneider, 1975) and peanut cell cultures (Van Huystee, 1977). Non-chlorophyllous callus of these plants contains only limited ALAD and PBGase activity.

In an attempt to test for the possible precursor of ALA, 14C-Iabelled glycine, aketo-glutarate and glutamate were fed to illuminated G-callus and the incorporation of 14C into Chi was examined (Table 1). The callus cells poorly incorporated 14C into Chi from [U-14C]a-ketoglutarate and [3,4-14C]glutamate. Not only were all the tested compounds respired, but the one which was respired the best, [U-14C]a-ketoglutarate, was the most ineffective donor of 14C to ALA. This finding suggests that a



permeability barrier is not responsible for the differences in the labelling ability of the tested compounds and that the labelling pattern is due to a lack of activity of the enzyme(s) involved in synthesizing ALA from these precursors. This explanation was further tested by the incorporation experiment with the chloroplasts prepared from N ingluba leaves and callus cells (unpublished data). In all the tested chloroplasts, appreciable quantities of [2-14C]glycine was incorporated into ALA, whereas little or no incorporation of labelled a-ketoglutarate and glutamate was found. There was some indications of an active system for the conversion of glycine to ALA in N ingluba callus chloroplast.

Incorporation of carbon from a-ketoglutarate or glutamate into ALA is considered to be evidence for the participation of the C-S pathway, while the incorporation from glycine is considered to be evidence for the ALA synthetase route (<<classical pathway») (Beale, 1978; Neuberger, 1980). Recently it was reported that both pathway are present in the plant kingdom (Beale, 1978; Schneider, 1971); in bacteria and algae, ALA production preferentially occurs via glycine and succinyl-CoA, and in higher plants also via glutamate. So the question arises which pathway operates preferentially in callus cells derived from higher plants. It seems reasonable that callus cells are organisms that possesses both pathways for ALA biosynthesis, and which operate preferentially according to physiological conditions. Under the experimental conditions employed, in N ingluba callus the ALA synthetase route operated more effectively than the C-S pathway.

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Metabolism of Chlorophyll in Higher Plants V. Chlorophyll Biosynthesis in Nicotiana Callus

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